DESCRIPTION (the applicant's description verbatim): Utilizing a combination of genetic engineering in mouse model systems and miniaturized physiological technology, the proposed studies will test two principal hypotheses concerning MLC2v function: 1) MLC2a cannot functionally substitute for MLC2v in ventricular cardiac muscle; 2) Phosphorylation of MLC2v plays an important role in ventricular papillary muscle and cardiac function, and impairment of MLC2v phosphorylation will result in FHC. The overall goals of the proposal are to understand the functional differences between two cardiac MLC2 isoforms, MLC2v and MLC2a and the functional role of MLC2v phosphorylation. We hope to gain insight into mechanisms by which mutations in MLC2v cause cardiomyopathy and the roles of environment, physical state, and genetic background in the incomplete penetrance and variable phenotype of this disease. We will achieve these goals by creating several gene targeted mouse lines through knock-in, and site-specific mutagenesis followed by comprehensive histological, biochemical, and physiological analysis of the resulting cardiac phenotypes. Accordingly, the Specific Aims are: 1. To investigate the functional equivalence of MLC2a and MLC2v by examining the ability of MLC2a to rescue the C2v null mutant phenotype. This will be performed by knocking the MLC2a cDNA into the MLC2v endogenous locus thereby deleting MLC2v and leaving MILC2a under the control of the endogenous MLC2v promoter. 2. To understand the functional significance of MLC2v phosphorylation and to determine whether elimination of MLC2v phosphorylation is sufficient to induce a form of hypertrophic cardiomyopathy. Mouse lines will be generated in which the phosphorylation site(s) (Ser 15 or Ser 15 plus Ser 14) of the endogenous MLC2v gene have been mutated to Ala. 3. To understand the mechanism by which MLC2v mutations cause familial hypertrophic cardiomyopathy (FHC) with middle left ventricular chamber thickening. A mouse model will be created by introducing a Glu22Lys mutation into the ALC2v gene (A Glu22Lys mutation in human MLC2v causes familial hypertrophic cardiomyopathy). For each mouse model proposed in Specific Aims 1-3, comprehensive physiological assessment of cardiac function, and detailed biochemical and biophysical analyses of resulting cardiac muscle phenotypes will be performed.